The present invention relates to DNA sequences which, on the codogenic strand, code plant debranching enzymes whose transcripts formed in transgenic plants code new proteins with the enzymatic activity of debranching enzymes which in transgenic plants reduce the degree of branching of amylopectin starch. The invention also relates to DNA sequences which on the codogenic strand code plant debranching enzymes whose transcripts formed in transgenic plants prevent the synthesis of proteins with the enzymatic activity of debranching enzymes, which in transgenic plants increases the degree of branching of amylopectin starch, and also to recombinant plasmids on which these DNA sequences are localized and which can be introduced into plant cells and plants.
The invention also relates to a process for the production of plants changed by genetic engineering whose amylopectin starch is modified, and to the modified starch obtainable from these plants.
Polysaccharides such as starch are, along with oils, fats and proteins, essential renewable raw materials from plants. A decisive factor which stands in the way of the use of renewable raw materials is the lack of substances which precisely meet the requirements of the chemical industry in regard to form, structure, or other physico-chemical parameters. In order to make the application of renewable raw materials feasible in as many fields of use as possible, it is particularly important to achieve a great material diversity. In regard to polysaccharides, this means that, for example, as many different forms of starch must be provided as possible. This necessitates considering both strongly branched forms which are characterized by a high surface reactivity in their chemical properties, and mildly branched types which are distinguished by a high uniformity of structure. Uniformity of structure is an important prerequisite for highly efficient reaction control during chemical syntheses.
Although starch is a polymer comprising chemically uniform basic components, the glucose molecules, it is a complex mixture of very different molecule forms which differ in respect to their degree of polymerization and the occurrence of branchings of the glucose chains. Starch is therefore not a uniform raw material. In particular, a distinction is drawn between amylose starch, an essentially unbranched polymer comprising xcex1-1,4 glycosidically linked glucose molecules, and amylopectin starch, which for its part is a complex mixture of differently branched glucose chains. The branchings come about through the occurrence of additional xcex1-1,6 glycosidic linkages. In typical plants for starch production, such as, for example, maize or potato, the two forms of starch occur in a ratio of roughly 25 parts amylose to 75 parts amylopectin.
In regard to the uniformity of a basic substance, such as starch, for its application in the industrial sector, plants are needed which, for example, contain only the component amylopectin or plants which contain only the component amylose. In regard to the versatility of the raw material starch, plants are needed which show forms of amylopectin with differently marked branching. There is thus a great interest in enzymes of the starch metabolism which can modify the degree of branching of the starch molecules, or in gene sequences which can be used for genetically changing plants so as to be able to synthesize different forms of starch in plants.
It is already known that for certain plant species, for example maize, plant types which contain only amylopectin can be produced by mutagenesis in which individual genes of the plant are inactivated. For potato, a genotype which forms no amylose was likewise produced by chemical mutagenesis with a haploid line (Hovenkamp-Hermelink et al., 1987, Theor. Appl. Genet. 75:217-221). Haploid lines, or the homozygotic diploid or tetraploid lines developed from them are not usable in agriculture, however. The mutagenesis technique is not applicable to the agriculturally interesting heterozygotically tetraploid lines, as inactivation of all copies of a gene is technically not possible because of the presence of four different genotype copies. It is known from Visser et al. (1991, Mol. Gen. Genet. 225:289) that plant types which form substantially pure amylopectin starch can be produced by antisense inhibition of the gene for the starch granule-bound starch synthetase in potato.
A branching enzyme of the potato is known from WO 92/14827. This enzyme is known as the Q-enzyme of Solanum tuberosum. It is also known that, with the help of DNA sequences which contain the information for the branching enzyme of the potato described in WO 92/14827, transgenic plants can be produced in which the amylose/amylopectin ratio of the starch is changed.
While the occurrence of several Q-enzymes is known for other species, e.g. maize (Singh and Preiss, 1985, Plant Physiol. 79:34-40), it is not known whether, besides the branching enzyme of the potato known from WO 92/14827, other enzymes are involved in the synthesis of branched starch in potato.
Besides the Q-enzymes which introduce branchings into starch molecules, enzymes occur in plants which dissolve branchings. These proteins, also known as debranching enzymes, are divided into three groups according to substrate specificity. The pullulanases, which besides pullulane also use amylopectin, occur in microorganisms, e.g. Klebsiella, and plants. In plants, they are also called R-enzymes. The isoamylases, which do not work with pullulane, but do with glycogen and amylopectin, likewise occur in microorganisms and plants. An isoamylase of maize is described by Manners and Rowe (1969, Carbohydr. Res. 9:107), and Ishizaki et al. (1983, Agric. Biol. Chem. 47:771-779) describe an isoamylase of potato. The amylo-1,6-glucosidases are described in mammals and yeasts and use limiting dextrins as substrates.
Besides five endo- and two exoamylases, Li et al. (1992, Plant Physiol. 98:1277-1284) detected only one debranching enzyme of the pullulanase type in sugar beet. This enzyme, which has a size of ca. 100 kD and a pH optimum of 5.5, is localized in the chloroplast. Ludwig et al. (1984, Plant Physiol. 74:856-861) describe a debranching enzyme from spinach which uses pullulane as a substrate but which displays an activity three times lower upon reaction with amylopectin.
In the case of the agriculturally important starch-storing cultivated plant the potato, the activity of a debranching enzyme was investigated in 1951 by Hobson et al. (1951, J. Chem. Soc. 1451). It was demonstrated that the corresponding enzyme, unlike the Q-enzyme, does not possess a chain extending activity and merely hydrolyses xcex1-1,6 glycosidic bonds. However, it was possible neither to characterize the enzyme more precisely nor to the describe DNA sequences which code a protein with the enzymatic activity of a debranching enzyme.
To date, no DNA sequences are known which code a protein with the enzymatic activity of a debranching enzyme from plants which, upon introduction into the plant genome, change the metabolism of the plant in such a way that the degree of branching of the amylopectin starch is increased or reduced.
The object of the present invention is to provide DNA sequences which code debranching enzymes on the codogenic strand, plasmids with which these DNA sequences can be introduced into plant cells or plants, plant cells from which whole plants can be regenerated and plants which make possible the production of amylopectin starch with an increased or reduced degree of branching.
There are now described the identification and purification of debranching enzymes and also peptide sequences of these enzymes and their use for the description of DNA sequences which in transgenic plants form transcripts which code proteins with the enzymatic activity of debranching enzymes, or which in transgenic plants form transcripts which prevent the synthesis of proteins with the enzymatic activity of debranching enzymes and plasmids and plant cells for the production of these transgenic plants.
Also described are transgenic plants which contain DNA sequences which code proteins with the enzymatic activity of debranching enzymes which reduce the degree of branching of amylopectin starch and transgenic plants which contain DNA sequences which prevent the synthesis of proteins with the enzymatic activity of debranching enzymes, which increases the degree of branching of amylopectin starch.
a) first, proteins with the activity of a debranching enzyme are purified to homogeneity (see example 1),
b) peptide sequences are established from the purified enzyme by protein sequencing (see example 2),
c) these peptide sequences are used for the cloning of cDNA sequences from a cDNA library, both immunological and molecular genetic methods being used (see examples 3 and 4) and/or
d) these peptide sequences are used for the cloning of genomic DNA sequences from a genomic library with the help of molecular biological methods (example 5) and finally
e) the DNA sequences from c) and/or d) are introduced into plasmids which make possible a transformation of plant cells and the regeneration of transgenic plants (see example 6).
The DNA sequences described by way of example in the examples with reference to the potato (S. tuberosum) code for a plant debranching enzyme which modifies the degree of branching of amylopectin starch naturally contained in plants in that the degree of branching of the amylopectin starch is increased or reduced as required.
Protein sequences of debranching enzymes with at least one of the following sequences are coded from the codogenic DNA sequences:
Transgenic plants with an increased or reduced degree of branching of amylopectin starch can be produced via a process which is characterized by the following steps:
a) Production of a DNA sequence with the following part-sequences:
i) a promoter which is active in plants and ensures the formation of a RNA in proposed target tissues or target cells,
ii) a DNA sequence which allows the transcription of RNA which, in transgenic plants, codes a new protein sequence with the enzymatic activity of a debranching enzyme or which allows the transcription of a RNA which in transgenic plants prevents the synthesis of a protein with the enzymatic activity of a debranching enzyme,
iii) if necessary, a 3xe2x80x2-non-translated sequence which in plant cells leads to the ending of transcription and to the addition of poly-A radicals to the 3xe2x80x2-end of the RNA,
b) transfer and incorporation of the DNA sequence into the genome of a plant cell, preferably using recombinant plasmids; and
c) regeneration of intact, whole plants from the transformed plant cells.
Preferably one, more or all of the protein sequences, SEQ ID NOs: 1-12, are contained in the protein sequence of the debranching enzyme named under ii). Recombinant plasmids according to process step b) contain the DNA sequences which on the codogenic strands code plant debranching enzymes or fragments thereof, whereby the transcripts derived from the DNA sequences in transgenic plants effect the synthesis of new proteins with the enzymatic activity of debranching enzymes which in the transgenic plants reduce the degree of branching of amylopectin starch or the transcripts derived from the DNA sequences in transgenic plants prevent the synthesis of endogenous proteins with the enzymatic activity of debranching enzymes, which in transgenic plants increases the degree of branching of amylopectin starch. The latter can be achieved through a compression or by anti-sense RNA (Inouye, 1988, Gene 72:25-34; and Flavell, 1994, Proc. Natl. Acad. Sci. USA 91:3490-3496).
The transgenic plants obtainable through the process with an increased or reduced degree of branching of amylopectin starch are also the subject of the invention. Plants to which the process is applied are useful plants such as e.g. maize, wheat and potato.
The invention also relates to proteins having the enzyme activity of a debranching enzyme, one or more of the sequences, SEQ ID NOs: 1-12 and a molecular weight between 50 kD and 150 kD, especially between 70 kD and 130 kD, above all between 90 kD and 110 kD. The proteins are from plants, such as S. tuberosum. 
For the identification of a new DNA sequence containing the information for the synthesis of a protein with the enzymatic activity of a debranching enzyme or for the suppression of the formation of an endogenous activity of debranching enzyme, protein extracts were obtained from plants, such as potato plants, by way of example. For the detection of the enzymatic activity of the debranching enzyme, as described in example 1, a colour test was used. When protein extracts of potato plants are separated in non-denaturing, amylopectin-containing polyacrylamide gels (PAAG), a protein with a starch-modifying activity can be detected by subsequent iodine dying. While unbranched amylose forms a blue-colored complex with iodine, amylopectin produces a reddish-violet colouring. The amylopectin-containing PAAGs which turn reddish-violet with iodine, at places at which a debranching activity is localized, a colour shift to a blue colouring of the gel occurs, as the branchings of the violet-colouring amylopectin are broken down by the enzyme.
By separating the protein from others with the help of progressive ammonium sulphate precipitation and subsequent affinity chromatography on immobilized xcex2-cyclodextrin, the protein is purified to homogeneity according to the invention. Peptide sequences are determined from the pure protein (see example 2). As a result, peptide sequences of a plant debranching enzyme are accessible for the first time. The peptide sequences of the debranching enzyme show, in individual areas, a certain homology to microbial debranching enzymes, but this is not true of all domains of the protein. The new debranching enzyme from S. tuberosum thus represents a previously unknown type of debranching enzyme.
The peptide sequences of the debranching enzyme serve according to the invention to identify DNA sequences in plants which code a peptide with the activity of a debranching enzyme. Immunological processes can be applied (see example 3) or molecular genetic methods are used (see examples 4 and 5).
After the DNA sequences which code a new debranching enzyme are identified, they can be multiplied in bacteria by cloning into vector plasmids. Examples of vectors are pBR322, pUC-series, m13mp-series etc. The DNA sequence which codes the new debranching enzyme can be provided with linkers which permit a simple recloning into other plasmids. For the purpose of introduction into plants (see example 6), binary plasmids which contain a replication signal, for example, for Escherichia coli and for Agrobacterium tumefaciens can be used preferably, but not exclusively. If these binary plasmids contain T-DNA elements, a transfer of the DNA sequence of a new debranching enzyme into the genome of dicotyledonous plants is particularly simple. Other methods are available however, for example, transformation with the help of ballistic processes which are used for the transformation of monocotyledons (cf. Potrykus, 1991, Ann. Rev. Plant Mol. Biol. Plant Physiol. 42:205-225).
To ensure an expression of the transferred transgene in genetically changed plants, the cDNA sequence of the new debranching enzyme is fused to a promoter sequence. All the promoters which are active in plants come into consideration in principle. Preferably promoters which are active in the starch-storing organs of the plants to be transformed are used. Thus, in the case of maize, it is the maize granules, whereas in the case of the potato, it is the tubers.
The tuber-specific B33 promoter (Rocha-Sosa et al., 1989, EMBO J. 8:23-29) can be used in particular, but not exclusively, for the transformation of the potato. For the stabilization of the RNA formed by the transgene, a termination and polyadenylation signal is also appended if necessary to the DNA sequence coding the debranching enzyme. This can be, for example, the termination signal of the octopine synthase gene from A. tumefaciens. 
By fusing a promoter, a DNA sequence and, if necessary, a termination signal, constructs are formed which are integrated into suitable plasmids for transformation of plants. These recombinant plasmids are also the subject of the present invention. The recombinant plasmids are used for the transformation of plant cells from which whole plants can be regenerated. These plant cells which contain the DNA sequences according to the invention are also the subject of the invention. The recombinant plasmids can also be used for the identification of nucleic acid sequences which code debranching enzymes.
As a result of the transfer of a DNA sequence which consists of promoter, coding region of a new debranching enzyme and termination/polyadenylation signal, a transgenic plant is produced in which RNA is formed which can serve as a matrix for the synthesis of a new debranching enzyme, or which, through interaction with an endogenous mRNA of a debranching enzyme, suppresses its synthesis. The type of RNA which is transcribed by the transgene depends on the orientation of the DNA sequence of the new debranching enzyme relative to the promoter. If the 5xe2x80x2 end of the DNA sequence of the new debranching enzyme is fused to the 3xe2x80x2 end of the promoter, a translatable mRNA is formed which serves as a matrix for the synthesis of a protein with the enzymatic activity of a new debranching enzyme. If, on the other hand, the 3xe2x80x2 end of the DNA sequence of the new debranching enzyme is fused to the 3xe2x80x2 end of the promoter, an antisense RNA forms which suppresses the translatability of the endogenous mRNA of the debranching enzyme.
In the first case, there is an additional enzymatic activity of a debranching enzyme in the plant. The result of this is that the degree of branching of the amylopectin formed by the transgenic plant is reduced. A starch thereby becomes accessible which, compared with the naturally occurring type, is distinguished by a more markedly ordered space structure and an increased uniformity, which has favorable consequences, for the film-formation properties in particular.
In the second case, an endogenous enzymatic activity of a debranching enzyme is suppressed. This leads to the formation of markedly branched starch in transgenic plants. Markedly branched amylopectin has a particularly large surface and is thereby suitable as copolymer to a particular extent. A marked degree of branching also leads to an improvement in the solubility of the amylopectin in water. This property is very favorable for certain technical applications. Potato is particularly suitable for the production of markedly branched amylopectin while exploiting the DNA sequences, according to the invention, of the new debranching enzyme, but the application of the invention is not limited to potato.
The modified starch formed in the transgenic plants can be isolated from the plants or from the plant cells with commonly used methods and processed after purification for the production of foodstuffs and industrial products. The DNA sequences which code for a debranching enzyme can also be used for the isolation of homologous cDNA or of genornic sequences from other plant species, using standard methods.